A physician specializing in Infectious Diseases, Dr. Heffernan returned to UCSD in August, 1988, entering a post-doctoral research fellowship. Since that time, he has worked with Dr. Guiney on a plasmid encoded serum resistance locus (rck) that is distinct from previously described serum resistance determinants. The rck locus was originally described on the virulence plasmid of Salmonella typhimurium, but he has found and mapped sequences homologous to rck in virulence plasmids of other Salmonella serotypes, including S. dublin. He has also determined that pADE016 (which contains the rck locus) confers serum resistance to a variety of E. coli and Salmonella, independent of LPS composition, and has sequenced the rck fragment. rck encodes an outer-membrane protein and this proposal is to explore the mechanisms by which the rck gene product specifies resistance to complement mediated killing. The proposed research will determine the rck gene responsible for the serum resistance phenotype, using a combination of deletion analysis, and random and site-directed mutagenesis. The rck associated outer membrane protein will then be overproduced isolated, and purified, in order to sequence the N-terminal regions to confirm our genetic analysis, and to use to immunize rabbits. Rck anti- serum will be used to further elucidate the nature of rck induced serum resistance by analyzing the interactions of rck protein with complement components on bacterial surfaces. The role of rck in native virulence plasmids will also be studied. Rck expression from plasmid containing and cured isogenic strains will be examined, and rck expression will be altered by site-directed mutagenesis. The effect of these mutations on serum resistance and on virulence in animals will be determined. The academic and research environment at UCSD is extremely conductive to the development of independent research skills in basic science. The sponsor, Dr. Guiney, has extensive experience in the molecular biology and pathogenesis of Salmonella. Along with Dr. Guiney, Drs. Sharon Reed and Joshua Fierer form a close collaborative research and training group, with expertise in complement evasion by micro-organisms and in bacterial virulence. This group should provide a wealth of opportunities to explore new techniques and provide additional training in the molecular genetics of bacterial pathogens.